This proposal contains plans for detailed biochemical and genetic studies of the interaction of transfer RNA with three selected proteins. (i) Glutaminyl-tRNA synthetase, a key enzyme in the pathway of Gln-tRNA(Glm) formation found in Gram-negative eubacteria and in eukaryotic cytoplasm, acylates tRNA with glutamine. Mutant enzymes with relaxed discrimination against tRNA have been isolated in E. coli, and the structure of the wild-type and mutant enzymes in complex with the tRNA is known at near atomic resolution. (ii) Glutamyl-tRNA reductase, the first enzyme in porphyrin biosynthesis in certain bacteria and chloroplasts, catalyzes the reduction of glutamate to glutamate-l-semialdehyde and requires a specific tRNA as cofactor. Thus, it is a key enzyme in heme synthesis in bacteria and chlorophyll synthesis in plants. (iii) Glu-tRNA(Glm) aminotransferase, a key enzyme in a different pathway of Gln-tRNA(Glm) formation, rectifies the incorrect aminoacylation of rRNA(Glm) which takes place during normal protein synthesis in Gram-positive eubacteria, archaebacteria and organelles. Our aim is to elucidate the basis for the specific interaction of transfer RNA with these enzymes in the hope of discovering possible homologies in the structures of proteins which interact with transfer RNA. These enzymes are 'hybrid' proteins; they contain motifs responsible for specific tRNA binding and a catalytic domain for different enzymatic functions (e.g., reduction, amidation, aminoacylation). Apart from defining the basis for specific RNA:protein interactions, this work will shed light on the role of transfer RNA in maintaining the accuracy of protein biosynthesis, on the involvement of this RNA molecule in intermediary metabolism, and on the evolution of aminoacyl-tRNA synthetases. The studies will be carried out in E. coli and Bacillus subtilis.